At the endoplasmic reticulum (ER) membrane, nascent prion protein (PrP) can be synthesized in both fully translocated and transmembrane forms, as a result of the action of Translocation Accessory Factors (TrAFs). The relevance of these observations has recently become apparent with the demonstration of spontaneous neurodegenerative disease in both mice and humans expressing PrP mutations that favor synthesis and export of transmembrane PrP from the ER. Some evidence suggests that the expression of neurodegeneration -associated transmembrane PrP may be a default outcome in the absence of TrAF activity. The investigation of topological regulation during PrP biogenesis is crucial for an understanding the molecular pathogenesis of these genetic prion diseases. Here we propose to dissect the mechanisms by which PrP biogenesis affects PrP topology disease. Here we propose to dissect the mechanism by which PrP biogenesis affects PrP topology, resulting in an transmembrane form that leads to neurodegeneration. Mutations in PrP structural domains will be systematically correlated to effects on topological regulation. Trafficking of selected mutants will be studied in transfected cells. Transgenic mouse model systems will be developed for those mutants with the most interesting phenotypes. Protein-protein interactions between the translocation machinery and nascent PrP will be identified and correlated with the pathway of neurodegeneration. Through this work, the pathway of PrP biogenesis, as it relates to a newly recognized pathway of neurodegeneration, will be better understood in molecular terms. In the long run these studies should make possible novel approaches to manipulation of PrP topology and may contribute to prevention or treatment of prion diseases and other neurodegenerative disease.